Cell lines of hematopoietic cells have been useful tools in studying the growth and differentiation of immature blood cells. The promyelocytic cell line HL60, Collins, Nature, 298:629 (1982), and the erythroleukemia-derived cell lines HEL, Martin et al., Science, 216:1233 (1982), and K562, Loggin et al., Blood, 45:321 (1978), having phenotypes characterized by both platelet and erythrocyte properties, have been extensively studied, but are restricted in their capacity to differentiate in response to chemical and biological inducers. Otherleukemia-derived cell lines which are dependent upon recombinant growth factors have recently been reported and represent several maturation stages of myeloid or lymphocytic lineages. Ihle, International J. Cell Cloning, 7:68 (1989).
Factor-dependent colonies of erythrocytes have been obtained from fresh human bone marrow (Perrine et al., Biochem. Biophys. Res. Comm., 148:694 (1987)) and peripheral blood (Rovera et al., Anal. Biochem., 85:506 (1987)) in semi-solid culture systems. Even though these biological systems have contributed significantly to the understanding of erythropoiesis, they too have been limited in their capacity to represent the complete differentiation pathway beginning with a noncommited progenitor and ending with a mature hemoglobin-containing erythrocyte.
Factor-dependent cell lines with properties of megakaryocytes have recently been reported. Avanzi et al., Brit. J. Haem., 69:359 (1988). Most of the characterization of megakaryocytes, both past and present, has been achieved using either the transformed cell lines or megakaryocytes freshly isolated from bone marrow before and after short-term cultures in either suspension or in colony assays. The conventional techniques used to characterize these cells have been morphology in culture, surface and cytoplasmic phenotype, as determined by monoclonal and polyclonal antibodies, biochemical studies of the synthesis of lineage-specific proteins or ultrastructure by electron microscopy. However, a well-defined phenotype for megakaryocytes has not yet been established. The cell lines as well as the uncultured early leukemic blasts have a mixed phenotype in that both megakaryocyte and erythroid markers appear within the same population of cells. This may be attributed to several factors, including (i) lineage infidelity of leukemic cells due to aberrant differentiation, (ii) wider distribution of markers on other lineages, or presence of markers before occurrence of lineage commitment and (iii) cellular expression of markers of more than one lineage for a brief period of time during the process of differentiation, before loss of properties not associated with the more mature stages. One possibility arising from these alternatives is the existence of a common precursor for erythroid and megakaryocytic lineages. Dessypris et al., Brit. J. Haem., 65:265 (1987). If such a cell exists, one must look for the earliest events associated with the event of lineage commitment, which would be at the level of gene expression. The standard methodology used for cell characterization reflects very late events subsequent to protein synthesis and does not provide crucial information regarding gene activation. Molecular techniques have not been widely used in the study of hematopoiesis due to either the limited numbers of cultured fresh cells, or the differentiation restriction of the transformed cell lines. One exception is the study of the globin gene switch in the committed erythroid cell.
Molecular and cellular biological methods have been used to characterize the globin genes and the natural course of the globin gene switch in which fetal gamma globin is replaced by adult beta globin. This gene switch occurs in development during the first year of life and also during adult bone marrow hematopoiesis. Much information has been derived from studies on human cell lines induced to produce minute amounts of adult hemoglobin, as well as on the MEL murine virus-induced erythroleukemia cell line, but globin gene modulation in these systems occurs in a renewing population of cells. T. Rutherford et al., Nature 280, 164 (1979); P. Martin et al., Science, 216, 1233 (1982); and M. Kaku et al., Blood, 64, 314 (1984). Although fresh erythroid cells have been grown in colony assays, the progeny of the progenitor cells are virtually inaccessible for the study of molecular events of gene expression. Molecular Basis of Blood Diseases: Hemoglobin Switching, p. 74, J. Dyson, ed., Saunders (1987).
There is a need for a biologically-relevant in vitro human system in which (i) cells can undergo a natural progression of erythroid maturation including the production of fetal and then adult hemoglobin; (ii) proliferation can be selectively and biologically inhibited before the induction of erythropoiesis and during the gene switch; and (iii) sufficient numbers of cells are readily available at select times during maturation for molecular analyses.